Expression And Analysis Of CcCBFs Genes In Camphora Camphora And Functional Verification In Model Plants

Low temperature is one of the world's impact on plant growth which is a serious natural disaster.The plants are subject to chilling injury in the process of development and growth,leading to declining agricultural yields and frozen large quantities of forestry trees,seriously which can also cause death.And which caused by the loss of up to hundreds of billions of dollars every year.Therefore,improving the plant the cold resistance has a great significance to reduce the loss of resistance in agriculture and forestry.The methods of traditional breeding in plants have made great progress,but the method of gene engineering does have an advantage in short period,quick increases in plant breeding,which has occupied an important position.The researchers originally just consider one single functional gene into the plant on the process of resistance in plant using the method of gene engineering that people,but the plant resistance gene is not associated with only one gene,and may involve multiple simultaneous expression of genes,so single gene resistance is not significant.Some transcription factors and upstream regulation the CBF/DREB1(CRT/DRE binding factor)gene was used in genetic operation is a kind of regulation of plant cold stress related gene expression of transcription factor.It can transfer the temperature signal perception upstream,in the low temperature induced rapid synthesis,and the related gene promoter upstream of the corresponding cis acting element of CRT/DRE(C-repeat/dehydration responsive element)combined to promote gene transcription downstream resistancein in the cold,then the synthesis of sugar,proline and stable membrane proteins and other substances,improve the cold resistance of plants.A large number of researchers have found that the resistance of transgenic CBF gene on the plants increased,and a new way to improve plant cold resistance by means of genetic engineering.This study firstly screened reference genes of Cinnamomum camphora under different abiotic stresses.Followed by laboratory cloned CcCBFs(CcCBFa,CcCBFb,CcCBFc,CcCBFd)genes of C.camphora.as the research foundation,to investigate the expression pattern of CcCBFs genes in different under abiotic stress.Finally,to discuss the function of CcCBFs genes in Arabidopsis and tobacco,which lays a foundation for the further development of transgenic camphor.The main results are as follows:1.The camphor seedlings was analyzed,the expression level and expression stability of two candidate reference genes CcACTc and CcEF1? in C.camphora under various abiotic stresses were analyzed by qRT-PCR technique and BestKeeper software,respectively.The results indicated that,CcEF1? was relatively stable under low temperature treatment,and it demonstrated higher stability under other abiotic stress,such as drought,high salinity and ABA treatment compared to CcEF1?.This shows that the CcEF1? is the better reference gene for quantitative analysis of stress-related genes in C.camphora.2.Camphor clones EL6 plantlets were kept in laboratory as experimental materials.The results showed that the expression level of CcCBFc gene was higher than that CcCBFa,CcCBFb and CcCBFd at low temperature stress,and the expression of CcCBFd gene was still higher than that of control group after 72 h stress.In response to drought stress,CcCBFa and CcCBFb genes more quickly,while the CcCBFc and CcCBFd genes high expression.Under the high salt stress,the expression of CcCBFb and CcCBFc genes significantly.To sum up,CcCBFc and CcCBFd genes was more sensitive under different abiotic stresses3.Arabidopsis thaliana seeds of CcCBFs genes of T2 were obtained by inflorescence infection,and then analyze the long in different abiotic stress seedling germination rate and average root.Results showed that at low temperature and drought stress,the germination rate of transgenic CcCBFd gene in Arabidopsis thaliana and average root length were higher than those of the wild type,then for CcCBFc transgenic Arabidopsis thaliana.Under high salt stress,the germination rate of transgenic CcCBFs are low,but the germination rate of CcCBFd transgenic Arabidopsis thaliana has a slight advantage.Thus,under low temperature,drought and high salt stress,the transgenic plants of CcCBFd gene have higher stress resistance4.To obtain transgenic tobacco expressing CcCBFs genes of C.camphora through genetic transformation of Agrobacterium mediated method(T1 transgenic seeds oftransgenic tobacco with CcCBFd gene have just been harvested at present),the different abiotic stress determination of proline,soluble sugar and MDA content.The results showed that under low temperature stress,transgenic tobacco have different degrees of ability to resist low temperature stress,the CcCBFc transgenic tobacco has a strong cold resistance,followed by CcCBFa transgenic tobacco;drought resistance of CcCBFa transgenic tobacco is best and CcCBFb transgenic tobacco is secondary in drought stress;CcCBFa and CcCBFc gene in tobacco salt tolerance were higher under high salt stress.Thus,transgenic tobacco resistant to CcCBFa and CcCBFc genes showed stronger resistance under low temperature and high salinity stress,and drought resistance of tobacco with CcCBFa and CcCBFb genes was stronger.